Project Summary Engineered CRISPR systems have the potential to transform the treatment of inherited diseases via genome editing-based cures. Nonetheless, safe, effective, and target-tissue-specific delivery of CRISPR effector proteins and their small RNA guides represents a major barrier to clinical application. Because of the central importance of the small RNA guides, CRISPR?s clinical development could benefit from technologies developed for earlier generations of nucleic acid therapeutics such as siRNAs and antisense oligonucleotides. Two critical realizations have led to a surge of recent successes with these therapeutic modalities: (i) the importance of complete chemical modification (i.e., the removal or modification of 100% of 2?-OH groups) to confer metabolic stability and suppress immune system activation without nanoparticle formulation; and (ii) the utility of appended chemical conjugates to tune biodistribution properties and engage cell-surface components that facilitate uptake. These principles should enable the safe, effective delivery of CRISPR guides, either pre- loaded into their protein effectors [ribonucleoprotein (RNP) delivery] or administered in tandem with mRNAs or viral vectors that encode the effector protein. In the latter case, uncoupling guide RNA delivery from vector- based effector delivery promises additional benefits including: (1) improved guide-target multiplexing (in parallel or in series), (2) flexibility to clear viral genomes via self-targeting in the target tissue after the desired editing has occurred, or from ancillary tissues (to limit prolonged effector expression that induces off-target editing and immune responses), (3) the ability to more precisely focus tissue-specific editing through orthogonal targeting moieties for guide and effector, and (4) the liberation of vector genomic capacity for other purposes. Despite the clinical promise of fully modified, conjugated, self-delivering CRISPR guide RNAs, they remain underdeveloped. The goal of this proposal is to establish and optimize such guide RNAs as a new therapeutic modality in CRISPR genome editing, in conjunction with multiple routes of effector protein delivery. We have identified a framework for complete modification and stabilization of guide RNAs for the most commonly deployed CRISPR effector (SpyCas9). We have also developed chemical modifications that increase the potency and stability of DNA donors that direct precise repairs, as needed for many diseases. We propose to combine our nucleic acid modification framework with our established roster of targeted, hydrophobic, endosomolytic, and pharmacokinetics-modifying conjugates to enable the safe and effective delivery of the genome editing machinery to tissues of the central nervous system, muscle and kidney in vivo, first in mice and then in pigs. We will pursue this goal with SpyCas9 and with three other editing effectors with complementary attributes. In addition, we will build in the capability to co-deliver repair templates with our modified guides, in both RNP and viral co-delivery formats, to enable precise gene repairs in vivo. Successful completion of the proposed work will realize important new delivery capabilities for therapeutic genome editing.